VZV对Schwann细胞PrPC 和EPO表达的影响及钴胺素干预机制研究

Herpes zoster associated neuralgia is typical of neuropathic pain, patients is very painful. Methylcobalamin has obvious analgesic effect on it, to have the important meaning to explore the mechanism of treatment of pain. For patients with blister fluid detected local TNF-1 decreased obviously after the treatment, while EPO increased significantly. Subcutaneous plexus of Schwann cells (SCs) exist, the SCs is activated to produce PrPC and EPO has neuroprotective effect. We found that the use of VZV infected rats SCs, SCs TNF- alpha and EPO all had high expression, Mecobalamine can down regulate TNF- alpha, the up-regulated expression of HIF-1 alpha and EPO. We speculate that, Mecobalamin is regulated by SCs PrPC, the stability of HIF-1 alpha, promoting the secretion of EPO. To test this hypothesis, the selection of primary cultured human SCs, step by step construction of PrPC, PHD2 or CCS gene silencing SCs model, were co cultured with VZV cells, and tissue and animal models were prepared, from the molecular to explore multi level SCs in VZV stimulated the expression and mechanism of PrPC and EPO, regulating effect of mecobalamin on HIF-1 alpha /EPO and molecular mechanism, at the same time acquisition of skin tissue is verified. To lay the foundation for revealing the mechanism of cobalamin treatment of herpes zoster neuralgia.

带状疱疹神经痛是神经病理性疼痛，患者十分痛苦，而甲钴胺有明显治疗效果，探究其治痛机制有重要意义。对水疱液检测发现治疗后局部TNF-1α减少，而EPO升高。皮下神经丛中schwann细胞（SCs）可分泌EPO。对大鼠SCs研究发现，VZV刺激下TNF-α、HIF-1α和EPO都有一过性上调。甲钴胺干预可下调TNF-α，上调HIF-1α和EPO。我们推测，VZV激活的SCs可产生神经保护分子PrPC和EPO，甲钴胺通过调节SCs的PrPC，稳定HIF-1α，促进EPO的分泌。为验证此假说，选用人SCs，分步构建PrPC，PHD2或CCS基因沉默SCs模型，与VZV共培养，同时制备组织和动物模型，采集患者皮肤组织；采用分子生物学手段，从分子，细胞到组织多层面探究SCs在VZV刺激下PrPC和EPO的表达变化，甲钴胺对HIF-1α/EPO的调节及机制。为揭示钴胺素治疗带状疱疹神经痛的机制打下基础。

临床研究发现甲钴胺对带状疱疹神经痛有明显的治疗作用，探究其治痛机制有重要意义。VZV激活施万细胞后，可迅速上调TNF-α等促炎因子。TNF-α转换酶（TACE）可切割跨膜TNF-α释放，并可切割TNFR1使其脱落。而TACE可被朊蛋白（PrPC）所调节。SC中有PrPC高表达，并沿着轴突分布。PrPC又可调节抗氧化酶SOD的活性。本项目探究VZV对hSC中PrPC，抗氧化酶SOD，TACE和TNFR1表达及活性的影响，及甲钴胺（MeB12）的调节作用。.结果发现VZV感染后细胞Prnp的mRNA表达明显上调，蛋白的表达未明显增加，但PrPC的单糖基化条带密度明显增加，上清与沉淀中的PrPC聚糖比例与对照组比较有明显变化，总聚糖比分别为1:1.5和1:2.6，提示VZV感染后PrPC稳定性下降。TACE染色颗粒明显变小，TACE活性为对照组的36%，TNFR1表达明显增多，上清中有少量sTNFR1。相应的SOD活性明显下降，MDA水平明显升高。.感染后加药组Prnp mRNA表达上调3.7倍，PrPC向单糖基化迁移的比例较感染组明显减少，沉淀中的聚糖有明显增加，总聚糖比为1:2.4，提示MeB12提高了PrPC的稳定性。与感染组比较，TACE染色颗粒变大，且TACE的活性提高，TNFR1表达减少，sTNFR1水平明显增加。相应地SOD活性明显增高，MDA水平明显下降。.Prnp-siRNA转染细胞感染VZV再加药处理，发现对TACE和TNFR1的表达及活性，sTNFR1含量的影响不明显。提示MeB12是通过稳定PrPC，调节TACE、TNFR1的表达及活性。.研究证实VZV感染可改变hSC中PrPC的糖基化特征，使PrPC稳定性下降，继而影响其抗氧化能力，TACE的活性和TNFR1的脱落。而MeB12可调节PrPC的糖基化特征，提高PrPC的稳定性，提高hSC的抗氧化能力，调节TACE活性，促进TNFR1的脱落，促进质膜上的TNFR1脱落来控制细胞对TNFα的敏感性，从而增强hSC的抗TNF-α神经毒性能力，达到神经保护的作用。.摸索了3通道同时评估亚急性HON患者三叉神经眼支的感觉神经传导。发现围刺配合MeB12注射可调节局部NSE、SP和 CGRP 释放，使得IL-2与IL-4比例趋于平衡。.MeB12干预的机制之一是调节了施万细胞PrPC翻译后糖基化修饰过程。